Alzheimer's disease (AD) and diabetes are currently common and frequently-occurring disease. Especially, their incidence is increasing as the old people growing. So the prevention and cure of these diseases are urgent problems to be solved today.
Current drugs therapy for AD are unlikely to revolutionize the treatment of AD due to their limitation of the mere symptomatic relief or severe adverse effects. The current drugs used for diabetes mainly are insulin and other orally hypoglycemic drugs. The inconveniency for long-term use and toxicity limited their wide applications and there are actually no effective drugs for type 2 diabetes. The recent study revealed that amyloid-beta (A β) and amylin (IAAP) are the basic molecules of AD and type 2 diabetes. The fibrillogenesis and subsequently increased free oxidative radicals are the triggers of these diseases, which gives rise to the fact that the fibril formation inhibitor of amyloid-beta and amylin becomes the perspective for the cure of these diseases.
Alginates are a family of linear polysaccharide produced by brown seaweeds and some bacterial species belonging to the genera Pseudomonas and Azotobacter. These polymers are composed of two hexuronic acids, β-D-mannuronic acid (ManA) and α-L-guluronic acid (GulA), linked by 1-4 bonds. Alginate belongs to high polymers with molecular weight of several 104 to 106 with very abundant source. The polymer alginate has been used for a variety of industrial purposes, e.g. as a stabilizing, thickening and gelling agent in food production and pharmaceutical applications. Recent developments revealed that alginate is an important biological active compound. Its application as a drug is largely limited by its huge molecular weight. So the oligosaccharide degraded from alginate by different methods is very valuable for glycochemistry, glycobiology, glycoengineering and saccharide-based drugs. The currently-used methods to degrade alginate include enzymatic, physical and chemical treatments, yet the finding of specific lyases has limited the application by enzymatic method. Physical method, which is usually used in combination with other method, couldn't get oligosaccharide with the minimal molecular weight of 50,000. The chemical degraded method used for polysaccharides include acidic hydrolysis method and hydrogen peroxide degraded method. The former method is limited by its capacity to get oligosaccharides with weight less than 4000 when conducted in normal temperature and pressure.
Contents of the Invention
Through deep studies by the inventors, it is found that an alginate oligosaccharide with molecular weight below 4000 can be obtained by acid hydrolysis at high temperature and pressure, and its derivatives whose reduced terminal in position 1 is carboxyl radical can be prepared in the presence of oxidants. The invention is completed on the above basis.
The invention provides an alginate oligosaccharide and its derivatives with low molecular weight, or pharmaceutically-acceptable salts thereof, and provides the process for preparing the same. The invention also provides a medicament for the prophylaxis and treatment of AD and diabetes with the above-mentioned low molecular alginate oligosaccharide or its derivatives, or pharmaceutically-acceptable salts thereof.
The invention relates to an alginate oligosaccharide and its derivatives represented by formula (I) or pharmaceutically-acceptable salts thereof, the above-mentioned oligosaccharide is composed of β-D-mannuronic acid linked by 1,4 glycosidic bonds,

wherein, n represents an integer of 0 or any number ranging from 1 to 19.
In the present invention, an example of the above alginate oligosaccharide derivatives is a compound represented by formula (II), in which the reduced terminal in position 1 is carboxyl radical,

wherein, n represents a integer of 0 or 1 to 19.
In above formula (I) and (II), preferable n is 2 to 10, and more preferable n is 4 to 8. The reason why better biological effects of tetrasaccharide over dodecasaccharide (preferably, hexasaccharide to decasaccharide) remains unclear, which may be caused by its liability to be recognized and accepted by cells.
The alginate oligosaccharide derivatives also include, for example, the derivatives, of which part of the hydroxyl groups in mannuronic acid are sulfated.
For example, the pharmaceutically-acceptable salts of the above alginate oligosaccharide and its derivatives can be salts of sodium, potassium, calcium, magnesium salts and the like. The sodium salts are preferred. The pharmaceutically-acceptable salts can be prepared by the conventional methods.
The invention also relates to a process for preparing above alginate oligosaccharide and its derivatives, characterized by the fact that an alginate solution is reacted for about 2 to 6 h in an autoclave at pH 2˜6 and the temperature of about 100˜120° C.; after which the value of pH is adjusted to about 7. The oxidative product is obtained by the addition of an oxidant to the alginate oligosaccharide solution.
In a preferred embodiment of the invention, a 0.5˜5% sodium alginate solution is heated to 110° C. for 4 h in an autoclave at pH 4. After the reaction is accomplished, the reactant is sucked out and cooled, then pH is adjusted to 7 by adding NaOH solution. With stirring, the filtrate is slowly poured in to industrial alcohol which is 4 times as the volume of the filtrate, and stayed overnight to allow precipitation. The alcohol precipitate is filtered off with suction to dryness, and is dehydrated by washing with absolute ethanol. A white filter cake is obtained and dried in an oven at 60° C. to give a crude alginate oligosaccharide. The crude alginate oligosaccharide is formulated to a 10% solution, and is precipitated with 95% ethanol solution. The precipitate washed with absolute ethanol, dried and formulated to a 5% solution. The solution is filtered through a 3 μm membrane to remove impurity, then desalted on a Bio-Gel-P6 column (1.6×180 cm) with 0.2 mol/L NH4HCO3 as mobile phase and then the product is obtained collected. The elute is detected by the sulfate-carbazole method, collected components including sugar, concentrated under reduced pressure and desalted, and lyophilized to give the alginate oligosaccharide.
The preparations of the derivatives represented by formula (II) is are as follows: an oxidant is added and reacted for 15 min to 2 h at the temperature of 100˜120° C. after the step when the above alginate solution reacting for about 2 to 6 h in an autoclave at pH 2˜6 and the temperature of about 100˜120° C. In an embodiment of the invention, 25 ml of 5% copper sulfate is added to 50 ml of 10% NaOH (aq), mixed immediately, and immediately added 40 ml of 5% alginate oligosaccharide solution. The resultant mixture is heated in a boiling water bath until no more brick red precipitate is generated. The mixture is centrifuged to remove precipitate. Some supernatant is taken out and added to 10% NaOH (aq) and 5% copper sulfate according to the above ratio to check any generation of brick red precipitate. If negative, the supernatant is added to industrial alcohol which is 4 times the volume to of the supernatant, and stayed overnight to allow precipitation. The precipitate is filtered off with suction to dryness, dehydrated with absolute ethanol repeatedly and dried in an oven at 60° C. The separation is preceded according to the same separation method of the alginate oligosaccharide of formula (I).
The invention also provides a pharmaceutical composition containing an effective amount of such alginate oligosaccharide or its derivatives, or pharmaceutically-acceptable salts thereof and pharmaceutically-acceptable carriers.
The pharmaceutical composition can be a medicament for the prophylaxis and treatment of AD.
Furthermore, the pharmaceutical composition can be an amyloid-β protein fibrils forming inhibitor and fibrils disaggregating promoter.
The pharmaceutical composition also can be a medicament for the prophylaxis and treatment of diabetes.
Moreover, the pharmaceutical composition can be used as pancreatic islet amyloid protein fibrils forming inhibitor and islet amyloid polypeptide inhibitor. In view of the current difficulty of lacking effective medicines for the prophylaxis and treatment of AD and diabetes, it is especially important that the alginate oligosaccharide of the invention is used in the manufacture of a medicament for the prophylaxis and treatment of AD and diabetes.